Chapter 6 - The mechanism of transcription in bacteria. In this chapter we will focus on the basic mechanism of transcription. We will begin with RNA polymerase, the enzyme that catalyzes transcription. We will also look at the interaction between RNA polymerase and DNA. | Molecular Biology Fourth Edition Chapter 6 The Mechanism of Transcription in Bacteria Lecture PowerPoint to accompany Robert F. Weaver Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. RNA Polymerase Structure By 1969 SDS-PAGE of RNA polymerase from E. coli had shown several subunits 2 very large subunits are b (150 kD) and b’ (160 kD) Sigma (s) at 70 kD Alpha (a) at 40 kD – 2 copies present in holoenzyme Omega (w) at 10 kD Was not clearly visible in SDS-PAGE, but seen in other experiments Not required for cell viability or in vivo enzyme activity Appears to play a role in enzyme assembly 6- Sigma as a Specificity Factor Core enzyme without the s subunit could not transcribe viral DNA, yet had no problems with highly nicked calf thymus DNA With s subunit, the holoenzyme worked equally well on both types of DNA 6- Testing Transcription Core enzyme transcribes both DNA strands Without s-subunit the core enzyme has basic transcribing | Molecular Biology Fourth Edition Chapter 6 The Mechanism of Transcription in Bacteria Lecture PowerPoint to accompany Robert F. Weaver Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. RNA Polymerase Structure By 1969 SDS-PAGE of RNA polymerase from E. coli had shown several subunits 2 very large subunits are b (150 kD) and b’ (160 kD) Sigma (s) at 70 kD Alpha (a) at 40 kD – 2 copies present in holoenzyme Omega (w) at 10 kD Was not clearly visible in SDS-PAGE, but seen in other experiments Not required for cell viability or in vivo enzyme activity Appears to play a role in enzyme assembly 6- Sigma as a Specificity Factor Core enzyme without the s subunit could not transcribe viral DNA, yet had no problems with highly nicked calf thymus DNA With s subunit, the holoenzyme worked equally well on both types of DNA 6- Testing Transcription Core enzyme transcribes both DNA strands Without s-subunit the core enzyme has basic transcribing ability but lacks specificity 6- Promoters Nicks and gaps are good sites for RNA polymerase to bind nonspecifically Presence of the s-subunit permitted recognition of authentic RNA polymerase binding sites Polymerase binding sites are called promoters Transcription that begins at promoters is specific, directed by the s-subunit 6- Binding of RNA Polymerase to Promoters How tightly does core enzyme v. holoenzyme bind DNA? Experiment measures binding of DNA to enzyme using nitrocellulose filters Holoenzyme binds filters tightly Core enzyme binding is more transient 6- Temperature and RNA Polymerase Binding As temperature is lowered, the binding of RNA polymerase to DNA decreases dramatically Higher temperature promotes DNA melting 6- RNA Polymerase Binding Hinkle and Chamberlin proposed: RNA polymerase holoenzyme binds DNA loosely at first Binds at promoter initially Scans along the DNA until it finds one Complex with holoenzyme loosely bound at the promoter is a .
